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Abstract Exploring the structural and electrical properties of DNA origami nanowires is an important endeavor for the advancement of DNA nanotechnology and DNA nanoelectronics. Highly conductive DNA origami nanowires are a desirable target for creating low‐cost self‐assembled nanoelectronic devices and circuits. In this work, the structure‐dependent electrical conductance of DNA origami nanowires is investigated. A silicon nitride (Si₃N₄) on silicon semiconductor chip with gold electrodes was used for collecting electrical conductance measurements of DNA origami nanowires, which are found to be an order of magnitude less electrically resistive on Si₃N₄substrates treated with a monolayer of hexamethyldisilazane (HMDS) (∼10¹³ohms) than on native Si₃N₄substrates without HMDS (∼10¹⁴ohms). Atomic force microscopy (AFM) measurements of the height of DNA origami nanowires on mica and Si₃N₄substrates reveal that DNA origami nanowires are ∼1.6 nm taller on HMDS‐treated substrates than on the untreated ones indicating that the DNA origami nanowires undergo increased structural deformation when deposited onto untreated substrates, causing a decrease in electrical conductivity. This study highlights the importance of understanding and controlling the interface conditions that affect the structure of DNA and thereby affect the electrical conductance of DNA origami nanowires.